EP2029186A1 - Isolated nature-identical collagen - Google Patents
Isolated nature-identical collagenInfo
- Publication number
- EP2029186A1 EP2029186A1 EP07725658A EP07725658A EP2029186A1 EP 2029186 A1 EP2029186 A1 EP 2029186A1 EP 07725658 A EP07725658 A EP 07725658A EP 07725658 A EP07725658 A EP 07725658A EP 2029186 A1 EP2029186 A1 EP 2029186A1
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- EP
- European Patent Office
- Prior art keywords
- collagen
- biomatrix
- tissue
- solution
- cells
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0068—General culture methods using substrates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/24—Collagen
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
- C08L89/00—Compositions of proteins; Compositions of derivatives thereof
- C08L89/04—Products derived from waste materials, e.g. horn, hoof or hair
- C08L89/06—Products derived from waste materials, e.g. horn, hoof or hair derived from leather or skin, e.g. gelatin
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/50—Proteins
- C12N2533/54—Collagen; Gelatin
Definitions
- the present invention relates to isolated collagen, methods for producing and isolating the collagen from collagen-containing tissue, and the use of the isolated collagen in a biomatrix, as an in vitro test system, tissue replacement or organ replacement.
- Extracellular matrices or carrier structures for the cultivation of cells, so-called biomatrices, are usually produced from isolated collagen, ie matrix proteins isolated from collagen-containing tissue. Especially in regenerative medicine such biomatrices play an important role.
- the goal of tissue engineering is to support or replace diseased, failed or lost tissue or organ functions.
- tissue or organ structures are usually cultivated ex vivo by seeding and cultivating at least one tissue-specific cell type on provided carrier structures or biomatrices. Biomatrix and cultured cells form the tissue or organ equivalent or replacement.
- biomatrix For the realization of tissue-specific functions, it is necessary for the cells applied to the biomatrix to reorganize or re-form the matrix proteins. For successful cultivation of the cells on such carrier structures, it is therefore important that matrix proteins are present which have as far as possible native or nature-identical properties.
- Known biomatrices are usually made from collagen, which is extracted from freshly prepared collagen-containing tissue, such as skin or tendons, that is, isolated.
- a known extraction method is the extraction with aqueous acetic acid.
- acetic acid is used in a concentration of 0.1% up to 500 mmol / l.
- collagen-containing tissue for example rat tail tendons (RTT)
- RTT rat tail tendons
- it is stirred with acetic acid over a period of 20 hours to 7 days, usually at room temperature.
- the disadvantage is that due to the high viscosity of the collagen solution, the final concentration of collagen in the extract fraction is limited.
- a particular disadvantage is that the native structure of the extracted matrix proteins is attacked during the incubation due to acid hydrolysis by the acetic acid and due to enzymatic digestion by mostly existing proteinases and the matrix proteins are partially or completely denatured.
- the subsequent regeneration of the original native collagen structure and the restoration of the biological functions of the matrix proteins are therefore limited. If the matrix proteins isolated by means of acetic acid extraction and partially denatured are subsequently lyophilized, the properties of the collagen product reconstituted using the lyophilisate deviate greatly from native collagen; that its use for the cultivation of tissue-specific cells is largely limited. In addition, the biomatrices prepared from the matrix proteins isolated by means of acetic acid extraction have further disadvantages such as uncontrolled (usually one-dimensional, vertical) shrinkage.
- the technical problem underlying the present invention is to provide methods and means for producing, recovering or isolating collagen or matrix proteins from collagen-containing tissue, the isolated collagen having a higher proportion of matrix proteins having a nature-identical structure.
- An associated further technical problem is to provide improved biomatrices suitable for the production of organ or tissue substitutes or equivalents or in vitro test systems.
- An associated other technical problem is in vitro test systems for the analysis and diagnosis of infections as well as for the investigation and testing of or degenerate or genetically modified human or animal cells, diagnostics and therapeutics which overcome the disadvantages of the prior art described above.
- the underlying technical problem is essentially solved by a method for isolating collagen or collagen matrix proteins from collagen-containing tissue according to the characterizing part of claim 1.
- collagen-containing fibers are isolated from the collagen-containing tissue in a step (a) and in a subsequent step (b) the isolated collagen-containing fibers in an aqueous urea solution containing urea in a concentration of 5 to 15 mol / l, especially 7 to 12 mol / l, more preferably about 9 mol / l, whereby or whereby a collagen-containing fraction is released from the fibers and dissolved out.
- the separating of the dissolved collagen-containing fraction of fiber and tissue residues follows in a step (c), so that a collagen-containing aqueous solution is obtained.
- the urea is separated from the collagen-containing fraction and preferably in a further step (e) the collagen-containing fraction freed from urea is preferably renatured in buffer solution so that renatured isolated collagen is obtained in aqueous solution.
- the invention therefore provides for incubating isolated collagen-containing fibers substantially with a highly concentrated urea solution for a certain period of time, with the collagen matrix proteins being dissolved out of the tissue.
- the extraction according to the invention allows the isolation of predominantly nature-identical matrix proteins, which after separation of the urine Stoffs in aqueous buffer solution can be almost completely renatured.
- the circular dichroism spectroscopy (DC) of the collagen fraction isolated according to the invention detects the typical so-called "triple helices" structure of native collagen, those in the DC spectrogram particularly by a negative band between 217 and 227 nm and possibly a weaker negative band
- a UV spectroscopic examination (UV) of the collagen fraction isolated according to the invention also shows a characteristic amino acid signature of histidine at 213 nm, which likewise indicates an intact native protein structure
- Collagen matrix proteins without having to use the antibody tests known to be disadvantageous, which disadvantageously often show cross-reactions, are therefore also an object of the invention is a process for the isolation of collagen or collagen matrix proteins, wherein the concentration determination of the coll Agent by UV.
- the collagen isolated according to the invention can be used to obtain a biomatrix and from it a tissue or organ replacement or equivalent or an in vitro test system which no longer has the known disadvantages of, in particular one-dimensional, shrinkage.
- the proliferation of tissue-specific cells and the new synthesis of matrix by the cells from the biomatrix is improved or at least to the same extent as with biomatrices obtainable by known methods.
- the collagen-containing tissue used, from which the collagen-containing fibers are isolated in step (a), is isolated from rat tails. cock crave.
- the collagen-containing tissue is a, preferably acellularized, porcine small intestine.
- the urea solution is stirred with the collagen-containing fibers.
- the collagen-containing fibers are incubated with the urea solution for 12 to 36 hours, more preferably for about 24 hours, preferably with stirring, so that the collagen-containing fraction is dissolved out of the fibers particularly effectively.
- step (c) the dissolved collagen-containing fraction of fiber and tissue residues by mechanical separation processes in a conventional manner, preferably separated by centrifugation.
- step (c) is alternatively or additionally separated by filtration.
- step (c) it is expedient in step (c) to separate the dissolved collagen-containing fraction by fractionation by means of gel filtration and, if appropriate, to separate it. Subsequently, if appropriate, several of the separated collagen fractions can be reunited.
- the person skilled in the art can thus select the composition of the matrix proteins that is favorable and / or tissue-specific for the respective field of application.
- the isolated collagen-containing fraction or the collagen fractions separated by fractionation is preferably subjected to analytical methods.
- the characterization of the matrix proteins preferably takes place by means of DC. In a further preferred variant, the characterization takes place alternatively or additionally by UV. In a further preferred variant, the characterization takes place alternatively or additionally by ESI-MS / MS. In a further In a preferred variant, the characterization takes place alternatively or additionally by means of MALDI-TOF / TOF.
- the protein fractions can be separated and isolated in a manner known per se by one-dimensional or two-dimensional gel electrophoreses, isoelectric focussing and / or SDS-PAGE.
- a gradient dialysis is preferably carried out in step (d).
- the cold that is preferably at temperatures from 0 0 C to about 1O 0 C, preferably at about 4 ° C, and dialysed for a period of 4 to 12 days, preferably 7 days.
- it is dia lysed against water.
- the aqueous collagen solution thus prepared has a collagen concentration of about 3 to about 8 mg / L.
- the dialysed fractions are concentrated to a specific collagen content, preferably by a factor of 2 or 3.
- the concentrates obtained are particularly suitable for the subsequent separation in SDS-PAGE, for further characterization and for preparing boiling-resistant biomatrices.
- the collagen fraction is then renatured in the further step (e) in phosphate buffered saline (PBS) or similar media or buffer systems.
- PBS phosphate buffered saline
- the invention also provides an isolated collagen which can be prepared by means of the method according to the invention or with it will be produced.
- the aqueous collagen solution obtainable according to the invention contains a high proportion of undenatured, native collagen in an aqueous medium, especially a proportion of the total collagen in solution of> 50%, in particular>60%,>70%,>80%,> 90% or> 95 %, preferably> 99%.
- Such a collagen preferably has the characteristic triple helices "signature" of the triple helices structure (see above) in circular dichroism spectroscopy.
- the resulting collagen-containing fraction aqueous solution is lyophilized, so that a storable dry collagen preparation is obtained.
- the lyophilized collagen thus obtained is subsequently mixed with water or a suitable medium, an almost completely native three-dimensional structure of the collagen is advantageously produced. Destruction of the naturally occurring triple helix structure by lyophilization does not occur.
- a further subject of the invention is also a lyophilized collagen obtainable according to the invention.
- the aqueous collagen fraction is lyophilized in a manner known per se, so that a dry, storable collagen preparation is obtained.
- it is also possible to temporarily store the solution in a frozen state for example at -1O 0 C to -80 0 C, preferably at about -20 ° C.
- Another object of the invention is a process for the preparation of a biomatrix, which is characterized in that first the steps (a) to (e) of the method according to the invention are carried out, whereby an aqueous collagen solution or collagen-containing fraction is obtained, and in one further step (f) the resulting collagen solution with cell culture medium or similar chem in a ratio of 2: 1 to 1: 2, preferably 1: 1, are mixed to a collagen-containing matrix precursor solution.
- the collagen content of the solution is preferably 3 to 8 mg of collagen per ml of solution, more preferably 5 to 7 mg of collagen per ml of solution.
- the matrix precursor solution thus obtained preferably at elevated temperature, preferably at 21 0 C to 37 0 C, gelled to a collagen-containing biomatrix.
- the collagen gel is obtained by dissolving the lypophilized collagen preparations obtainable according to the invention.
- Another object of the invention is accordingly also a biomatrix which can be prepared by the aforementioned method or preferably produced therewith.
- a further subject of the invention is also a biomatrix which contains or preferably consists of the collagen isolated by means of the method according to the invention.
- the invention relates in particular to a, preferably gelatinous, biomatrix, for use in cultivation methods, for example for culturing cells of a specific tissue type or cells of a plurality of tissue types.
- the combination of biomatrix and cells cultured therein can be used to prepare an in vitro tissue or organ assay system.
- a “biomatrix” is understood as meaning a gel structure which contains collagen, cell culture medium, buffer and possibly serum Preferred cell culture media are DMEM (Dulbecco's Modified Eagle Medium) and M 199.
- a preferred buffer system is Hepes buffer. FCS) or human serum, especially autologous serum, the pH of the solution of cell culture medium, buffer and serum is in zugter execution 7.5 to 8.5, for example, 7.6 to 8.2, in particular 7.8.
- the biomatrix may contain other factors, for example growth factors, protease glycans, glucosaminoglycans, adhesives, antibiotics, selection agents and other extracellular matrix components.
- a cell culture medium, serum and buffer preferably several times (x times) concentrated, are precultivated with cells, preferably 1 ⁇ to 2 ⁇ 10 5 cells per ml, preferably 1 ⁇ 5 ⁇ 10 5 Cells per ml. Subsequently, for example in a ratio of 1: 2 to 2: 1, mixed with the collagen solution according to the invention in the cold at 0 to 10 0 C, in particular at 4 ° C, mixed.
- the mixing ratio (volume) of collagen solution to cell suspension (buffer, serum, cells, culture medium) is preferably 1: 1, with x-fold concentrated gel solution especially a volume ratio of (x-1): 1 collagen solution to gel solution is preferred.
- the suspension is pipetted into culture vessels and, after gelling at elevated temperature, preferably happens at 37 0 C, which within generally a few minutes, with medium overlaid (submerged culture).
- the biomatrix can be cultured for 2 days. Subsequently, additionally cells of other tissue types can be settled and cultivated thereon.
- a preferred embodiment of the invention comprises the cultivation of animal or human cells in a three-dimensional gel-like biomatrix for the multiplication of these cells and for the production of a three-dimensional animal or human in vitro tissue or organite test system.
- Results achieved with tissue or organ testing systems may be more meaningful than results obtained with animal experiments and may ensure better portability to humans.
- a subject matter of the invention is also a tissue equivalent or replacement which contains the biomatrix according to the invention as well as vital tissue cells, in particular tissue-specific cells, which are preferably cultivated in and on or in or on the biomatrix.
- An object of the invention is also an organ equivalent or replacement, containing the biomatrix according to the invention as well as vital tissue cells, in particular organ-specific cells, which are preferably cultured in and on or in and on the biomatrix.
- the invention encompasses the cultivation of human dermal fibroblasts in the biomatrix for the production of a dermis equivalent and epidermis equivalent three-dimensional human in vitro skin equivalent.
- “Culturing of cells” is a maintenance, preferably in vitro, of the tissue-typical life functions of cells, for example of fibroblasts, in a suitable environment, for example under the supply and removal of metabolic products and products, in particular also an increase in the cells .
- dermal fibroblasts are naturally understood to mean, in particular, dermis or genetically modified fibroblasts or their precursors.
- Fibroblasts are the precursors of dermal fibrocytes.
- the fibroblasts may be of animal or human origin; it may be tissue freshly isolated cells or cells of primary or genetically engineered or transformed cell lines such as WI-38, NIH / 3T3, MRC-5.
- the organic matrix contains the fibroblasts to be cultured and a collagen scaffold newly constituted from the collagen solution obtainable according to the invention, having a concentration of 2 to 5 mg, preferably 3.5 to 4.5 mg, of collagen per ml of biomatrix.
- the collagen scaffold is obtained from a solution according to the invention, preferably cell-free, of collagen, wherein the protein concentration of the collagen solution is preferably 5 to 7 mg / ml.
- fibroblast-containing biomatrix is the collagen solution in the cold, preferably at 4 0 C, with a cell solution or - suspension containing one, preferably five-fold concentrated cell culture medium, buffer, preferably HEPES buffer, serum, preferably fetal calf serum ( FCS), and chondroitin (4/6) sulfate, and fibroblasts, preferably at a concentration of about 1.5 x 10 5 / ml, and mixed well.
- This mixture is gelled, for example by raising the temperature to room temperature or 37 0 C in about 2 minutes. After gelling the gels, fibronectin is preferably added to the gels.
- Fibronectin mediates the binding of cells to macromolecules, such as collagen and attachment to neighboring cells.
- the subsequent culturing of the fibroblasts in the collagen gel is preferably carried out in submerged culture; this means that the cells are covered with a nutrient solution.
- the fibroblast-containing biomatrix is overlaid with cell culture medium and cultured at 37 ° C and 5% CO 2 under standard conditions in a conventional manner.
- the fibroblasts cultured in the biomatrix can be removed again from the biomatrix and, if appropriate, reintroduced into a biomatrix, the cells not losing their specific metabolic activities and their differentiation status after leaching out.
- the method according to the invention thus makes it possible to carry out intermediate cultivation of the fibroblasts in the biomatrix. With a small starting amount of fibroblasts, the method according to the invention therefore offers the advantage that sufficient cell material can be made available for the production of dermis equivalents and / or skin equivalents.
- a further advantageous embodiment of the invention provides that dermal fibroblasts, which are to be checked in terms of their function, their morphology and / or their differentiation status, are introduced into a three-dimensional biomatrix mentioned above, cultivated and / or checked thereafter.
- the invention therefore also relates to screening and diagnostic methods performed using dermal fibroblasts, wherein the fibroblasts can be cultured according to the method described above and / or subsequently investigated, for example for pharmacological, oncological, toxicological, physiological, morphological and / or or molecular biological parameters.
- this biomatrix contains a framework of human or animal collagen constituted by a collagen solution, ie tissue-typical matrix proteins.
- this capsule fibroblast gel is preferably subjected to a one- to two-day submerse culture.
- the dermal fibroblasts are cultivated in the three-dimensional biomatrix, as described above, so that subsequently a dermis equivalent can be obtained.
- a dermis equivalent is understood as meaning a connective tissue-like layer of collagen and fibroblasts. the one that largely corresponds to the native dermis.
- one to three days, preferably two days, after the above-described incubation of the gels, keratinocytes, skin stem cells or precursor cells of the keratinocytes, preferably precultured, undifferentiated keratinocyte stem cells, are sown on the gel from preferably human biopsy tissue, with KBM- Covered medium and subjected to one to three days Submers cultivation.
- Complete differentiation of the keratinocyte layers is achieved by an airlift culture in about 1.8 mmol / l CaCb-containing KBM medium (without hEGF and BPE).
- an "airlift culture” is understood as meaning a culture in which the level of the nutrient medium level is precisely matched to the height of the biomatrix, while the keratinocytes or cell layers formed by the keratinocytes are above the nutrient medium level and from the nutrient medium
- the culture is carried out at the boundary layer air-nutrient medium, whereby the cultures are supplied from below, by inserting the inserts from the microtiter plate with 24 wells into the wells of a 6-well microtiter plate, each with a diameter
- an airlift culture preferably 12 to 14 days, a typical dermis equivalent and epidermis equivalent skin-specific in vitro solid skin model is developed, which can be advantageously used for the test methods according to the invention.
- Keratinocytes or keratinocyte stem cells are then seeded on the fibroblast-containing biomatrix.
- keratinocytes is meant cells of the epidermis that form keratinizing squamous epithelium, or genetically engineered keratinocytes or their precursors that are of animal or human origin can. Since the formation of a well-differentiated epidermis with intact keratinization strongly depends on the proportion of basal stem cells in the keratinocytes used, the keratinocytes seeded on the collagen gel are preferably undifferentiated keratinocyte stem cells from human biopsy tissue, ie cytokeratin 19- or Integrin ß1 -positive basal stem cells. Preferably, these are pre-cultured cells, particularly preferably keratinocytes in the first or in the second cell passage.
- keratinocytes with a comparatively high proportion, for example 0.5%, 1%, 2%, 5%, 8% or 10% of the keratinocyte cell population, or comprising only undifferentiated stem cells are used.
- specific culture conditions including in particular a multi-day submerged culture and subsequent multi-day airlift culture of the biomatrix system, and specific culture media, the keratinocytes undergo differentiation into a multilayered epidermal layer.
- other cell types and / or other cells of other tissue types can be seeded on the biomatrix before, during or after seeding of the keratinocytes, for example immune system cells.
- the dermis equivalent is not subjected to any undefined shrinkage process in the course of the cultivation period.
- the invention therefore also relates to a skin-typical, three-dimensional, preferably human, in vitro skin equivalent, consisting of a dermis equivalent and an epidermis equivalent, and go for the production, cultivation and application of this skin equivalent and its components.
- Skin-specific full-skin models which are also referred to as in vitro skin equivalents, can be used as test skin, in particular in dermatology and in allergology, for their pharmacological effects to substances, for example potential drugs or cosmetics, or agents, such as light and heat , in particular irritation, toxicity and inflammatory effects, and to examine their tolerability.
- a further embodiment of the invention comprises the cultivation of intestinal fibroblasts with the biomatrix obtainable according to the invention for producing a three-dimensional in vitro human intestinal test system consisting of preferably Caco2 cells or intestinal epithelial cells or other human cell lines.
- Intestinal fibroblasts are naturally, especially in the intestinal tissue, occurring or genetically modified fibroblasts of animal or human origin or their progenitor cells.
- the collagen solution in a volume ratio of 1: 1, preferably at 4 0 C, with a solution also referred to as gel solution containing a preferably 2-fold concentrated cell culture medium, buffer, preferably Hepes buffer, and Serum, preferably 10% serum, and preferably 1, 5 x 10 5 / ml intestinal fibroblasts, in particular pre-cultured intestinal fibroblasts, mixed and mixed well.
- This mixture is gelled by raising the temperature to room temperature or 37 °.
- Subsequent cultivation of the intestinal fibroblasts in collagen gel is preferably carried out in submerged culture.
- the fibroblast-containing biomatrix is incubated at 37 ° C.
- a further subject of the invention is an in vitro test system comprising the biomatrix according to the invention and vital cells which are cultured in and on or in or on the biomatrix.
- the invention relates in particular to those in vitro test systems which can be used for the analysis and diagnosis of infections and / or diseases of the human or animal body caused by pathogenic and / or parasitic microorganisms, in vitro test systems for the analysis and diagnosis of degenerate human and animal Cells, in vitro test systems for the analysis and diagnosis of genetically modified human and animal cells and in vitro test systems for the testing and testing of anti-infectives and anti-tumor medicines, in particular cytostatics, and in vitro three-dimensional in vitro organ and tissue models, in particular susceptible to infection Weaved, such as intestines, liver, skin, cornea, trachea and mucous membranes.
- Cells to be examined are preferably cultured in the three-dimensional, gel-like, connective tissue-like biomatrix provided according to the invention and can multiply there.
- This biomatrix contains the cells to be cultivated in or on a collagen scaffold constituted by a collagen solution, ie tissue-specific matrix proteins.
- additional other cell types preferably other primary cells, can additionally be applied to the biomatrix.
- the cells preferentially contained in the biomatrix and the other cell types preferably applied to the biomatrix may undergo differentiation into a multi-layered three-dimensional animal tissue or organelle model.
- Such cocultivation of the animal in vitro Tissue or organ test system with a parasitic or pathogenic microorganism offers the opportunity to study both the infection process itself and the defense reaction of the corresponding organoid cell systems. For example, larger amounts of infected cell material and the pathogen itself can be recovered.
- the recovered material can then be further analyzed by conventional histological, biochemical, molecular biological or immunological methods, for example morphological changes of infected cells, the secretion of specific substances by the pathogen, such as toxins or proteins relevant for occurring resistance, or the release of specific substances by affected persons
- pathogen such as toxins or proteins relevant for occurring resistance
- a preferred embodiment of the invention also comprises the cocultivation of a three-dimensional in vitro tissue or organite test system according to the invention with a pathogenic or parasitic microorganism.
- pathogenic or parasitic microorganisms also referred to here as infectious agents, both eukaryotic and prokaryotic microorganisms, such as bacteria, fungi, protozoa, viroids, but also prions or viruses, understood that one Macroorganism, in particular a human or animal organism, and live in or on tissues of this organism and can lead to an infection of this organism, but do not necessarily have to lead to it
- coculture means a simultaneous, preferably maintaining in vitro, the life functions of animal cells and microorganisms in the same, for both suitable environment, for example, under supply and removal of metabolic factors and - products, in particular a simultaneous multiplication of cells and microorganisms.
- an in vitro tissue or organ test system according to the invention, especially in conjunction with a cocultivation method, the effect of chemical substances, in particular of anti-infectives, or of agents on the infection process or the growth of a pathogenic To study microorganism.
- agent is intended to encompass in particular chemical, biological or physical means, such as light or heat, which may exert a potential effect on living cells.
- a preferred embodiment of the invention involves the analysis of degenerate cells.
- the term "degenerate” encompasses all changes of a normal cell, for example cell polymorphism, anisocytosis, nuclear polymorphism, polychromasia, impaired nuclear-plasma relation and aneuploidy leading to disturbed differentiation or to a dedifferentiation and to a deregulated one
- the cells or malignant tumors in particular the tissues or organs mentioned above, are constructed from an in vitro gwew or organ test system in order to recover larger quantities of the degenerated cell material is obtained by conventional methods, for example histological see, biochemical, molecular biological or immunological methods, further analyzed to investigate the release of specific substances and to create transcriptional and expression profiles.
- the in vitro tissue or organ assay system constructed of degenerate cells is used to study the effect of drugs and potentially drug-suitable substances, in particular with regard to their ability to inhibit cell division.
- patient-specific degenerate cells are used to establish an in vitro tissue or organ assay system in order to investigate treatment options for the particular tumor disease of the patient.
- the review of genetically modified cells in particular the above-mentioned tissues and organs, provided.
- the term "genetically modified cells” encompasses all cells which have been manipulated with the aid of genetic engineering methods, whereby either foreign DNA and / or RNA has been introduced into the cell or the own DNA and / or RNA.
- a further subject of the invention is the use of the collagen according to the invention for the production of a tissue equivalent or replacement, for the preparation of an organ equivalent or replacement or for the production of an in vitro test system.
- FIG. 1 shows the histological presentation of fibroblast cultures after 45 days in / on a biomatrix provided according to the invention.
- tendons from rat tails are used as the collagen-containing tissue. All work is done under sterile conditions with sterile materials.
- the rat tails are superficially disinfected after storage at -2O 0 C with 70% alcohol.
- the skin of the rat tails is peeled off and the individual collagen fibers are withdrawn. If other starting tissues are used, any cells present may be gently removed by mechanical, enzymatic or chemical treatment.
- the collagen fibers are collected in phosphate-buffered saline (PBS) (pH 7.2), and in 70% alcohol for about 10 minutes superficially. then thoroughly washed with PBS. The weight of the fibers is determined.
- PBS phosphate-buffered saline
- the fibers are transferred into a highly concentrated urea solution; the final concentration is 9 mol / l. This approach is stirred for about 24 hours at about 4 ° C. Subsequently, the undissolved collagen parts are removed by centrifugation (1000 rpm, 1 hour, 8 ° C). The collagen is now in solution and not in fiber, scaffold or matrix form.
- the collagen solution is subjected to gel filtration in another batch.
- approximately 50 ml of the urea-containing collagen solution are applied after extraction to a 1.6 l column (diameter: 6 cm, height: 60 cm) with a Superose TM 12 (GE Healthcare) package and a flow rate of about 25 ml / h elutes. 100 fractions of 10 ml each are collected. Depending on the desired protein composition several fractions are reunited, others are discarded.
- the urea-free collagen fraction taken from the dialysis is lyophilized for preservation in a manner known per se.
- the structure of the collagen proteins obtainable according to the invention and fractionated by means of gel filtration and freed from urea by means of gradient dialysis against water were characterized biophysically.
- Circular dichroism spectroscopy is used to verify protein structures. This results in characteristic spectra of so-called “triple helices", which show a typical "signature” which is characterized by a small negative band between 217 and 227 nm and an optionally visible less intense negative band near 200 nm. These spectra are distinctly different from CD spectra of collagen proteins of random coil, ⁇ -helix, or ⁇ -sheet structures.
- the triple-helical structure is typical of collagen I 1, which dominates in aqueous solution, most of it of isolated collagen folds in water to the original native structure.
- collagen available according to the invention was lyophilized and then redissolved in water and fed to CD spectroscopy. It turns out that collagen solution prepared from lyophilized collagen also has the characteristic Has triple helix structure. Lyophilized collagen produced according to the invention shows a high solubility in water without irreparably destroying the native structure of the collagen. This property could not be achieved so far with collagen from natural sources.
- UV spectra of the isolated collagen fragmentation in the range of 190 to 320 nm were measured.
- the UV spectra confirm the analysis of the CD spectra.
- aqueous collagen solution obtainable by means of conventional acetic acid extraction was investigated.
- the collagen obtainable according to the invention shows characteristic amino acid residues in the UV spectrum. These include, above all, histidine at a wavelength of 213 nm. This advantageously permits the direct concentration determination of the collagen obtainable according to the invention by means of objective spectroscopic and photometric methods.
- fibroblasts were cultured on the matrix.
- matrices prepared by known methods acetic acid extraction
- fibroblasts a method known per se was used. Specifically, human donor skin (pre- puzien) with Dispase solution and then treated with trypsin solution. The epidermis layer was removed from the enzymatically pretreated pieces of tissue, the dermis was then comminuted with a scalpel and incubated for 30 to 45 minutes in collagenase type 4 (500 U / ml) at 37.degree. The mixture was then centrifuged at 1000 rpm for 5 minutes with suction of the supernatant. The pellets were resuspended in DMEM + 10% FCS and recentrifuged.
- the pellets were taken up in 2 ml of DMEM + 10% FCS and transferred to an uncoated cell culture flask. After culturing for one to two days, an additional 10 to 15 ml of DMEM + 10% FCS was added and further cultivated. Every three days, a media change was carried out. The culture was carried out in a steam-saturated incubator under conventional cell culture conditions (37 ° C and 5% CO 2 atmosphere).
- the fibroblasts are passaged and taken up in a gel casting solution (consisting of cell culture medium, serum and buffer) at a concentration of 1 ⁇ 10 4 cells / ml and are pipetted into a collagen solution obtainable by means of acetic acid extraction.
- a gel casting solution consisting of cell culture medium, serum and buffer
- the mixture was taken, mixed simultaneously and pipetted 500 ⁇ l each into a 24-well plate.
- the gels were incubated at 37 ° C. for 5 minutes in an incubator to allow gel gels to gel. After gelling the gels, they were mixed with about 1.5 ml / well DMEM + 10% FCS + 1% gentamycin and cultured at standard cell culture conditions. After one week, a severe shrinkage of the cultures was observed in the comparative batch. After a cultivation period of about 18 days, the exter- in the comparative batch due to excessive shrinkage.
- the invention obtainable according to biomatrix was initially profiled in the inventive approach, and subsequently to this, the cells according to passengers in a concentration of 1 x 10 4 cells / ml in gel casting as fibroblast suspension. Then, culturing was carried out in a manner known per se (see comparative experiment). After more than 40 days of culture it was confirmed by vital staining that the fibroblasts had grown well. By microscopic observation and histological discoloration (hematoxolin-iosin), this growth could be clearly observed and visualized even after 60 days. The histological staining also shows that the fibroblasts from the biomatrix have synthesized a new matrix (FIG. 1).
- Example 5 Preparation of a multilayer in vitro skin model
- a skin model consisting of human fibroblasts and primary keratinocytes was prepared.
- the human fibroblasts were recovered as in Example 4.
- the keratinocytes are obtained as follows: The epidermis separated from the enzymatically treated piece of tissue was subjected to a further trypsin treatment and then mechanically comminuted. The mechanically comminuted epidermis particles were picked up by a special keratinocyte medium and / or stopped with trypsin inhibitor. The cells are then centrifuged at 1000 rpm for 5 minutes, the supernatant is discarded and the pellets are carefully mixed with 2 ml of keratinocytes. Resuspended medium and transferred to a culture vessel. After about 4 hours, a change of media is performed under sterile conditions. The media change then takes place every two days. The cultivation is carried out in a manner known per se under standard cultivation conditions (see Example 4).
- the collagen solution obtainable according to the invention (see Example 1) and a so-called gel casting solution, consisting of cell culture medium, serum and buffer, were initially introduced and placed on ice.
- the fibroblasts are passaged and taken up in a concentration of about 1 ⁇ 10 4 cells / ml in the gel casting solution and immediately pipetted air-free into the collagen solution.
- the mixture is taken up and mixed at the same time.
- 500 ⁇ l each are pipetted into a 24-well plate with a special insert (Nunc). Thereafter, the gels are incubated at 37 ° C for 5 minutes in an incubator.
- gels After gels have been gelated, they are mixed with approximately 1.5 ml of DMEM + 10% FCS + 1% gentamycin per well and incubated overnight at 37.degree. The next day, the suction of the medium takes place. 25 ml of fibronectin are then pipetted onto the gel at a concentration of 50 ⁇ g / mg and the gel is again placed in the incubator for about 30 minutes.
- the keratinocytes are passaged from the abovementioned culture and pipetted onto the gels 1 ⁇ 10 5 cells / ml in 50 ⁇ l / BMBr + 5% FCS. Subsequently is incubated for 20 minutes at 37 0 C. Thereafter, the gels are further cultured with KMBr + 5% FCS in a Submers culture. In the following five days of cultivation, the FCS concentration is reduced from 5% to 0%. Thereafter, the further Cultivation of the preparations for 12 to 14 days in air-exposed environment.
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DE102006026591A DE102006026591B4 (en) | 2006-05-31 | 2006-05-31 | Method for isolating collagen from collagen-containing tissue |
PCT/EP2007/004767 WO2007137827A1 (en) | 2006-05-31 | 2007-05-30 | Isolated nature-identical collagen |
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US (1) | US8013121B2 (en) |
EP (1) | EP2029186B1 (en) |
JP (1) | JP2009538854A (en) |
AT (1) | ATE489974T1 (en) |
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DE102009059901A1 (en) * | 2009-12-21 | 2011-06-22 | Julius-Maximilians-Universität Würzburg, 97070 | Collagen fiber constructs for cruciate ligament replacement |
DE102011011092A1 (en) * | 2011-02-09 | 2012-08-23 | Amedrix Gmbh | Gelling collagen and means for providing it |
CN104017073A (en) * | 2014-06-18 | 2014-09-03 | 常州药物研究所有限公司 | Method for preparing collagen |
CN104017072A (en) * | 2014-06-18 | 2014-09-03 | 常州药物研究所有限公司 | Complete device for collagen purification |
CN104857561B (en) * | 2015-04-21 | 2018-04-06 | 北京湃生生物科技有限公司 | Bionical collagem membrane of high intensity and preparation method thereof |
CN106946987B (en) * | 2017-02-20 | 2020-02-28 | 福建农林大学 | High-concentration acid-soluble collagen solution and preparation method of water-soluble collagen solution |
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DE10062626B4 (en) | 2000-05-31 | 2005-10-20 | Fraunhofer Ges Forschung | infection models |
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WO2007137827A1 (en) | 2007-12-06 |
US8013121B2 (en) | 2011-09-06 |
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US20100216239A1 (en) | 2010-08-26 |
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DE502007005857D1 (en) | 2011-01-13 |
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